Abstract

Epstein Barr Virus driven post transplant lymphoproliferative disease (PTLD) is a
serious complication following either stem cell or solid organ transplantation (SOT),
occurring as a result of suppressed cellular immunity. Adoptive transfer of EBV
specific cytotoxic T cells (EBV-CTLs) is effective as both prophylaxis and treatment
for PTLD following stem cell transplantation, but is less effective when applied to
PTLD after SOT. It is likely that the continued pharmacologic immunosuppression
designed to prevent graft rejection compromises the function of infused EBV-CTLs.
To address this issue, we have generated calcineurin (CN) mutants that do not bind the
immunosuppressants Tacrolimus (FK506) and Cyclosporin A (CsA), therefore
conferring resistance to these CN inhibitors. A panel of 54 such mutants was designed,
generated and screened in a Luciferase expression assay, with identification of those
capable of resisting suppression with FK506, CsA or both. Subsequently, in assays
utilising both the Jurkat T cell line and primary human EBV-CTL lines, mutant CNa12
conferred resistance to CsA, mutant CNa22 conferred resistance to FK506 and mutant
CNb30 rendered cells resistant to both calcineurin inhibitors. EBV-CTL lines
transduced with a retroviral vector encoding these mutants retained the ability to both
proliferate and secrete normal levels of interferon-\gamma in the presence of therapeutic and
supratherapeutic levels of FK506 (CNa12), CsA (CNa22), or both (CNb30). The
cytotoxicity, phenotype and antigen dependence of EBV-CTL lines were unaffected
by expression of mutant CN. A xenogenic murine model of PTLD was established in
the RAG2^{-/-}/\gamma c^{-/-}/C5^{-/-} triple knockout SCID mouse strain and the ability of EBV-CTL
lines to induce tumour regression was examined. Administration of EBV-CTLs caused
regression of subcutaneous LCL tumours in vivo in some donors. This model will be
utilised in further investigations of transduced EBV-CTLs in vivo. The generation of
EBV-CTLs that are resistant to CN inhibitors should allow effective immunotherapy
in the SOT setting without risking rejection of the graft by reducing
immunosuppression. Additionally this represents a generic approach to improving
immunotherapy in the face of immunosuppression in other settings.

Type:

Thesis
(Doctoral)

Title:

Generation of calcineurin inhibitor
resistant EBV-CTLs for the
treatment of post transplant
lymphoma

Open access status:

An open access version is available from UCL Discovery

Language:

English

Additional information:

The abstract contains LaTex text. Please see the attached PDF for rendered equations.